Glaucoma is a chronic eye disease in which the pressure inside the eye increases and leads to damage to the optic nerve, and eventually causes blindness. In this disease, it is often necessary to use a multi-drug treatment system. There is a fixed combination of timolol maleate and brimonidine tartrate among the combination drugs in glaucoma treatment. Liposomes are one of the most important targeted drug delivery systems to eye tissue, which leads to improved drug permeability and durability in ocular tissue. In this study, thin layer hydration was used to make liposomal formulations containing timolol maleate (TM) and brimonidine tartrate (BT). After the necessary evaluations, one of the eight initial formulations was selected as an optimization formulation. Then, characteristics such as drug loading percentage, particle size, pH, zeta potential, and drug release were performed on the optimized formulation. The study of reducing intraocular pressure was performed on the optimized formulation. This study in total was performed on 18 rabbits in three groups. Hydroxypropyl methylcellulose (HPMC) polymer was injected into the anterior chamber to experimental induce glaucoma. The selected formulation was within the acceptable range of ocular products in terms of physical properties. HPMC polymer injection successfully induced glaucoma in the animal model, resulting in a 79% increase in intraocular pressure. The results showed that the liposomal formulation significantly reduced the intraocular pressure compared to the simple formulation of the aqueous solution, and both formulations were able to significantly reduce the intraocular pressure compared to the control group (P < 0.001). The results also showed that liposomal formulation has a therapeutic effect in reducing intraocular pressure. It seems that the selected liposomal formulation made by thin layer hydration can act as a suitable drug carrier to increase the effectiveness of the fixed combination of timolol maleate and brimonidine tartrate and be proposed as a new drug formulation for targeted and controlled drug delivery in the treatment of glaucoma.